Delivery of oligonucleotides, nucleic acids and nucleic acid analogs inside cells is a fundamental requirement for their therapeutic efficacy as antisense agents (Yakubov, 1989). The main problems with available antisense reagents are their low intracellular stability and inefficient cellular uptake (Stein, 1993). Furthermore, the high doses of antisense reagents required for therapeutic action lead often to toxic side effects. These drawbacks can be circumvented by applying modified nuclease resistant oligonucleotides (Bennet, 1998) or enhancing intracellular delivery of antisense oligonucleotides. Because of its hydrophobic interior, the lipid bilayer of the plasma membrane is a highly impermeable barrier to most polar and charged molecules (Alberts, 1989). Many antisense nucleic acids falter as potential drug candidates by not attaining effective pharmacokinetics, i.e. effective intracellular concentrations.
The antisense approach relies on delivery of specific nucleic acid or nucleic acid analog sequences to inhibit the expression or replication of DNA at the transcriptional level ("antigene"), or mRNA at the translational level ("antisense"). From the many studies on the antigene and antisense mechanisms of action, it is clear that cellular uptake and distribution are key to therapeutic action (Helene, 1990; Akhtar, 1992; Stein, 1993). The present invention achieves improvements in these important parameters.
In view of the limitations and deficiencies of conventional constructs of peptides and nucleic acid analogs, and inadequate methods for intracellular delivery across membranes, it is of interest to develop novel constructs with membrane-permeable properties. Constructs containing peptide and peptide analog sequences that are transported across lipid membranes rapidly and in high concentration are desirable. Nucleic acid analogs moieties of constructs which can be released after transport and interact with target molecules are very desirable. Furthermore, it is desirable to provide cleavable linkers between the peptide and nucleic acid analogs moieties of the constructs.
Nucleic acid analogs which can hybridize to target polynucleotides are especially desirable. Hydrophobic, uncharged nucleic acid analogs are preferable as nucleic acid analogs moieties. Efficient methods for synthesis and purification of constructs are desirable. The invention herein provides for solutions for some of the limitations and deficiencies in transport and targetting of molecules into cells via optimized constructs and methods.